Project Summary The molecular and cellular interactions that orchestrate neural crest migration to ensure proper vertebrate head development have yet to be fully elucidated. Upon delamination from the neural tube, cranial neural crest cells destined to form the bone, cartilage and neurons of the face and neck are sculpted by intrinsic neural tube and local microenvironmental signals onto stereotypical pathways. What is unclear is how microenvironmental signals confine neural crest cells to distinct migratory pathways. Furthermore, neural crest-derived cancers such as melanoma and neuroblastoma have limited therapeutic agents to prevent their uncontrolled metastatic invasion. Thus, information about the factors that inhibit neural crest and neural crest-derived cancer cell invasion has the potential for widespread impact in preventing birth defects and cancer metastasis. We pioneered the in vivo visualization of embryonic chick neural crest cell migration using state-of-the-art 2- photon time-lapse microscopy, in ovo culture and quantitative cell behavior analyses. In this study, we propose to examine the biological function of candidate genes we discovered by microarray analysis to inhibit neural crest migration. By screening candidate gene expression patterns for their correlation with cranial neural crest cell free zones and using time-lapse microscopy to analyze cell behaviors in response to candidate proteins in stripe assays, we will rapidly converge on a short list of inhibitory factors. Furthermore, candidate proteins will be evaluated for their ability to inhibit human melanoma and neuroblastoma cell invasion. The success of this proposal will significantly advance our understanding of molecules that control neural crest cell migration. This information will provide a foundation for a future mechanistic study of the in vivo role of these factors to confine cranial neural crest cell migration and suggest signaling pathways to examine related to the metastatic control of neural crest-derived cancers.